Division of Hematology-Oncology, Department of Pediatrics, The University of Texas Southwestern Medical Center, Dallas, Texas, USA.
PLoS One. 2013 Aug 5;8(8):e70371. doi: 10.1371/journal.pone.0070371. Print 2013.
Recent studies show that Arf, a bona fide tumor suppressor, also plays an essential role during mouse eye development. Tgfβ is required for Arf promoter activation in developing mouse eyes, and its capacity to induce Arf depends on Smads 2/3 as well as p38 Mapk. Substantial delay between activation of these pathways and increased Arf transcription imply that changes in the binding of additional transcription factors help orchestrate changes in Arf expression. Focusing on proteins with putative DNA binding elements near the mouse Arf transcription start, we now show that Tgfβ induction of this gene correlated with decreased expression and DNA binding of C/ebpβ to the proximal Arf promoter. Ectopic expression of C/ebpβ in mouse embryo fibroblasts (MEFs) blocked Arf induction by Tgfβ. Although basal levels of Arf mRNA were increased by C/ebpβ loss in MEFs and in the developing eye, Tgfβ was still able to increase Arf, indicating that derepression was not the sole factor. Chromatin immunoprecipitation (ChIP) assay showed increased Sp1 binding to the Arf promotor at 24 and 48 hours after Tgfβ treatment, at which time points Arf expression was significantly induced by Tgfβ. Chemical inhibition of Sp1 and its knockdown by RNA interference blocked Arf induction by Tgfβ in MEFs. In summary, our results indicate that C/ebpβ and Sp1 are negative and positive Arf regulators that are influenced by Tgfβ.
最近的研究表明，Arf，一种真正的肿瘤抑制因子，在小鼠眼睛发育过程中也起着至关重要的作用。Tgfβ对于发育中的小鼠眼睛中 Arf 启动子的激活是必需的，并且其诱导 Arf 的能力依赖于 Smads 2/3 以及 p38 Mapk。这些途径的激活与 Arf 转录增加之间存在大量延迟，这意味着附加转录因子结合的变化有助于协调 Arf 表达的变化。我们专注于在小鼠 Arf 转录起始附近具有潜在 DNA 结合元件的蛋白质，现在表明，Tgfβ诱导该基因的表达与 C/ebpβ与近端 Arf 启动子的表达减少和 DNA 结合有关。在小鼠胚胎成纤维细胞（MEFs）中异位表达 C/ebpβ可阻断 Tgfβ诱导的 Arf 表达。尽管 MEFs 和发育中的眼睛中 C/ebpβ缺失会增加 Arf mRNA 的基础水平，但 Tgfβ仍能增加 Arf，表明去阻遏不是唯一因素。染色质免疫沉淀（ChIP）试验表明，在 Tgfβ处理后 24 和 48 小时，Sp1 与 Arf 启动子的结合增加，此时 Tgfβ 显著诱导 Arf 表达。Sp1 的化学抑制及其在 MEFs 中的 RNA 干扰敲低阻断了 Tgfβ诱导的 Arf 表达。总之，我们的结果表明，C/ebpβ和 Sp1 是受 Tgfβ影响的 Arf 的负调节因子和正调节因子。
